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Humanized monoclonal antibody and application thereof

A monoclonal antibody and humanized technology, applied in the field of medicine, can solve the problems of antibody concept change, affinity decrease, disappearance, etc.

Active Publication Date: 2018-01-16
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, such substitutions often lead to a change in the antibody's conformation, resulting in reduced or even lost affinity

Method used

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  • Humanized monoclonal antibody and application thereof
  • Humanized monoclonal antibody and application thereof
  • Humanized monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Humanized transformation of mouse monoclonal antibody 2A10G6

[0027] 2A10G6 murine antibody Fab design strategy is as follows:

[0028] PH promoter-BamH1-GP67-EcoR1-L chain-Hind3

[0029] P10 promoter-Sma1-HBM SP-Nhe1-H chain-His-Kpn1

[0030] The Fab heavy chain amino acid sequence (VH-CH1) of the mouse monoclonal antibody 2A10G6 is shown in SEQ ID NO: 7, the nucleotide sequence is shown in SEQ ID NO: 8, the mouse monoclonal antibody 2A10G6mFab light chain amino acid sequence (VL- CL) is shown in SEQ ID NO:9, and the nucleotide sequence is shown in SEQ ID NO:10.

[0031] Compare the amino acid sequences of the 2A10G6 heavy chain and light chain V regions with the reported human antibody sequences, and sort them according to the amino acid homology, select 10 known antibodies with the top rankings of the light and heavy chains, and then compare them according to the difference in CDR length. Whether the minimum, light and heavy chain interaction amino aci...

Embodiment 2

[0038] Example 2: Preparation and purification of monoclonal antibodies

[0039] The preparation methods of mouse antibody 2A10G6 and humanized antibody IgG h2A10G6 are as follows:

[0040] i) Plasmid construction: Insert the Fab gene sequence of the murine antibody 2A10G6 into the pFastBac-Dual vector, and add the name pFastBac-Dual-2AmFab behind the heavy chain gene. The humanized antibody IgG gene of the murine antibody 2A10G6 was inserted into the pFastBac-Dual vector, and a 6-histidine tag was added behind the gene, named pFastBac-Dual-2AhIgG. The recombinant plasmids were confirmed by PCR, enzyme digestion and sequencing to confirm that the inserted foreign fragments were completely correct.

[0041] ii) Preparation and purification of Fab fragment 2AmFab protein of murine monoclonal antibody 2A10G6 and humanized antibody h2A10G6 IgG protein:

[0042]First, the recombinant plasmid was transformed into DH10Bac competent cells, cultured overnight at 37°C, positive clones...

Embodiment 3

[0047] Example 3: Expression of Flavivirus Proteins

[0048] Expression of yellow fever E protein, dengue virus E protein, and West Nile fever E protein:

[0049] The DNA fragment of the extracellular region of YFV_E (the amino acid sequence is shown in SEQ ID NO: 11, and the nucleotide sequence is shown in SEQ ID NO: 12) was digested with NdeI and XhoI, and connected to the pET21a vector. The 3' end of the YFV_E protein coding region is connected with the coding sequence of 6 histidine tags (hexa-His-tag) and the translation stop codon. Then the ligation product was transformed into BL21 Escherichia coli competent cells. Single clones were inoculated into 40mL LB medium and cultured for 6-8 hours. Inoculate into 4L of LB medium and culture to OD at 37°C 600 =0.4-0.6, add IPTG to a final concentration of 1 mM, and continue culturing at 37°C for 4-6 hours. Inclusion bodies were harvested by refolding the inclusion bodies in the dilution method. The refolding solution (100m...

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Abstract

The present invention discloses a humanized monoclonal antibody and an application thereof, belonging to the technical field of medicine. In the invention, the humanized transformation is carried outon a rat monoclonal antibody 2A10G6, the rat monoclonal antibody 2A10G6 is expressed by baculovirus, and the humanized antibody h2A10G6 is obtained. The h2A10G6 antibody of the present invention has high affinity and neutralization activity against yellow fever virus, dengue fever and West Nile virus, and can be applied to clinical treatment and prevention of yellow fever virus, dengue virus and West Nile virus.

Description

technical field [0001] The invention relates to a humanized monoclonal antibody and its application, belonging to the technical field of medicine. Background technique [0002] Flaviviruses are a class of enveloped single-stranded positive-sense RNA viruses, including Zika (ZIKV), dengue fever (DENV), West Nile fever (WNV), yellow fever (YFV) and other viruses that are pathogenic to humans. These viruses are mainly transmitted by mosquitoes, and infection can have serious consequences for humans. Yellow fever virus infection mainly causes fever, muscle pain (lumbar pain and lower limb pain, etc.), headache, chills, loss of appetite, nausea and vomiting, severe high fever, jaundice abdominal pain accompanied by vomiting, and even death; dengue fever infection causes high fever, Headache, muscle, bone and joint pain and other clinical manifestations are one of the main causes of death in children in Southeast Asia; severe West Nile fever virus infection will cause aseptic men...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14
CPCY02A50/30
Inventor 高福秦成峰严景华邓永强戴连攀施一李晓峰仝舟路希山宋健
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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